Protein glycosylation is essential in all eukaryotes, from disease-causing protists such as malaria, to yeast and mammals. Secretory proteins are N-glycosylated, O- and C-mannosylated, and/or glycosylphosphatidylinositol (GPI)-anchored as they enter the lumen of the endoplasmic reticulum (ER). Yeast that cannot synthesize N- glycoproteins or GPI-proteins are inviable, and mice with the same defects die as embryos. Glycosylation is important in dengue and SARS-CoV-2 viral infections, and defects in glycosylation cause human disease. Thus, deficient O-mannosylation of dystroglycan is a cause of muscular dystrophy and GPI deficiency in hematopoietic human stem cells underlies the hemolytic disease paroxysmal nocturnal hemoglobinuria. Congenital Disorders of Glycosylation (CDGs) are severe inherited diseases with neurological symptoms. Protein glycosylation reactions require the glycolipids mannosyl- and glucosyl-phosphoryl dolichol (MPD, GPD) to act as sugar donors in the lumen of the ER. As these lipids are synthesized on the cytoplasmic side, they must be flipped across the ER membrane to function in the lumen, a process requiring specific transporters, termed scramblases, that have yet to be identified. Assays of the two scramblases in microsomes and reconstituted vesicles, using natural lipids and short-chain analogs as reporters, reveal that transport is bidirectional, ATP-independent, and highly structure specific, discriminating between structural isomers. We will identify the MPD and GPD scramblases using chemo-proteomic and bioinformatic approaches. Deploying novel photo-clickable probes synthesized by the Häner group (University of Bern) we will determine the MPD and GPD interactomes, that we hypothesize will include the scramblases. Our preliminary results validate this approach: the MPD probe functions in ER mannosylation and photo-identifies specific yeast microsomal proteins. Photo-adducted proteins will be identified by quantitative proteomics and tested for scramblase activity in our reconstitution-based assays. Promising candidates will be validated in vivo by evaluating phenotypes of yeast mutants. For GPD scramblase we will also identify candidates via phylogenetic profiling, a bioinformatics method for assignment of protein function. This approach complements the photo- identification strategy and has already yielded a list of GPD scramblase candidates for testing. This is a consequential proposal to discover critical players in ER protein glycosylation. Our extensive experience in studying scramblases puts us in a strong position to tackle this objective. We discovered the scramblase activity of Class A GPCRs and were the first to show lipid scrambling by a TMEM16 ion channel. We now deploy in silico, biochemical and biophysical methods to elucidate their mechanism. We will use this expertise in future work to reveal the molecular mechanism of structure-specific lipid scrambling mediated by the MPD and GPD scramblases that we...